Compositions for and methods of using herpes simplex virus glycoprotein D to suppress immune responses

ABSTRACT

Methods of delivering a desired polypeptide to an individual are disclosed. The methods comprise administering to the individual an immunogenic vector comprising a nucleic acid encoding the desired polypeptide operably linked to regulatory elements in combination with one or more of gD protein, a function fragment of gD protein, a nucleic acid encoding gD protein operably linked to regulatory elements, or a nucleic acid encoding a functional fragment of gD protein operably linked to regulatory elements. Compositions comprising an immunogenic vector that comprises a nucleic acid encoding the desired polypeptide operably linked to regulatory elements; and one or more of gD protein, a function fragment of gD protein, a nucleic acid encoding gD protein operably linked to regulatory elements, or a nucleic acid encoding a functional fragment of gD protein operably linked to regulatory elements are disclosed. Methods for inhibiting an undesirable immune response in an individual are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application PCT/US01/23648.

The present application claims priority to U.S. provisional applicationSer. No. 60/221,025 filed Jul. 27, 2000.

FIELD OF THE INVENTION

The present invention relates to methods of inhibiting anti-viralresponse, to improved gene delivery methods, to methods of treatingindividuals who have autoimmune diseases and to methods of treatingindividuals who have diseases, disorders and conditions associated withinflamation. The present invention relates to pharmaceuticalcompositions useful in such methods. The present invention relates toimproved gene therapy vectors and compositions which comprise HSV gD ora gene encoding the same, and methods of making and using the same. Thepresent invention relates to methods for delivering polypeptides toindividuals while inhibiting the cellular immune response against thevector which contains the nucleic acid encoding the desired polypeptide.

BACKGROUND OF THE INVENTION

One promise of gene therapy is the ability to correct genetic defectsresponsible for disease by the addition to an individual of functionalgenetic material as well as the ability to deliver therapeutic proteinsusing genetic material that encodes such proteins. There is a great dealof activity in the development of protocols for treating diseases anddisorders by administering a nucleic acid which codes for a polypeptidethat is either missing or defective in an individual. Another promise ofgene therapy is as an alternative and improved means to delivertherapeutically important proteins to individuals in need of suchproteins. The discovery of proteins with therapeutically importantfunctions has led to new treatments for many diseases and disorders andthe application of gene therapy to deliver such proteins is also thesubject of much interest.

Among the strategies for delivering genetic material, the use ofimmunogenic vectors, most commonly viral vectors, capable of infectingthe individual's cells is the one of the most widely employedmethodologies. Essentially, genetic material that encodes desiredproteins, whether they be functional forms of defective genesresponsible for disease or coding sequences for therapeutically usefulproteins, is incorporated into the genome of a vector which has theability to infect cells of the individual or otherwise deliver thegenetic material to cells of the individual.

Adenovirus, adenovirus associated virus (AAV), vaccinia virus, andsimian virus 4 (SV40) are just a few of the many viruses used to makeviral vectors for gene therapy. In some cases, the viral vectors areselected for their ability to infect specific tissue to which deliveryof the genetic material is desired. In some cases, the viral vectors areselected because they are attenuated and cause serious limitedinfections to the individual without significant pathology.

One of the major problems associated with gene therapy protocols thatemploy immunogenic vectors is that an immune response against the vectoris induced in the individual who is administered the vector. The immuneresponse targets the vector including cells which are infected by thevector. The destruction of cells which are infected by the vectorreduces the efficacy of the treatment. Further, immune responses inducedagainst the vectors limit the effectiveness of subsequent doses of thesame gene therapeutic composition or other gene therapeutic compositionswhich use the same vector because the immune system of the individualwill recognize the vector from the subsequent doses of the same genetherapeutic composition or other gene therapeutic compositions which usethe same vector and mount an immune response similar to the manner inwhich a vaccine protects the individual from subsequent exposure to apathogen.

There are two branches to the immune system. The humoral branch of theimmune system involves antibodies which are secreted by B lymphoid cellsand recognize specific antigens. Binding of antibodies to specificantigens inactivates the antigen. Antibodies may also bind to theantigen and activate other immune cells which destroy the bound antigen.

The cellular branch of the immune system involves specific cell typeswhich recognize and destroy cells which display “foreign” antigens.Cytotoxic T cells (also referred to as cytotoxic T lymphocytes or CTLs)are an example of cells in the cellular branch of the immune system.CTLs recognize fragments of peptides which are displayed on the plasmamembrane surface bound to major histocompatibility complex molecules(MHCs). Cells that display a peptide which is “foreign” elicit acellular immune response. Cytotoxic T cells then destroy the celldisplaying foreign peptide fragments.

When a patient undergoes an organ or cell transplant or a tissue graft,the patient's immune system will recognize the donor organ, cells ortissue as “foreign” and mount an immune response against the donororgan, cells or tissue. Immunosuppressive drugs are used to downmodulate the patient's immune response and prevent rejection.

In autoimmune disease, the immune system attacks “self” antigen. Someautoimmune diseases are T cell mediated. Examples of T cell mediatedautoimnmune diseases include Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Other autoimmune diseases are B cell mediated.Examples of B cell mediated autoimmune diseases are Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. In both types, an immune response is directed atthe body's own antigens. Autoimmune diseases may be treated bysuppressing immune responses.

There remains a need for improved gene therapy vectors, compositions andmethods which can be used to increase safety and efficacy. There remainsa need for improved gene therapy vectors, compositions and methods whichcan reduce or elimnate the immune response against the viral vectorwhich limits the ability to expose the individual to subsequent doses ofthe therapeutic or other therapeutics or vaccines employing the samevector. There is a further need for methods for suppressing immuneresponses associated with cell organ, cell and tissue transplants. Thereis a need for methods for delivering polypeptides to individuals whileinhibiting the cellular immune response against the vector which encodesthe desired polypeptide. There is a further need for methods formodulating immune responses associated with inflammatory and autoimmunediseases and disorders.

SUMMARY OF THE INVENTION

The present invention relates to methods of delivering a desiredpolypeptide to an individual. The methods comprise administering to theindividual an immunogenic vector comprising a nucleic acid encoding thedesired polypeptide operably linked to regulatory elements incombination with one or more of gD protein, a functional fragment of gDprotein, a nucleic acid encoding gD protein operably linked toregulatory elements, or a nucleic acid encoding a functional fragment ofgD protein operably linked to regulatory elements.

The present invention relates to compositions comprising an immunogenicvector that comprises a nucleic acid encoding the desired polypeptideoperably linked to regulatory elements; and one or more of gD protein, afunctional fragment of gD protein, a nucleic acid encoding gD proteinoperably linked to regulatory elements, or a nucleic acid encoding afunctional fragment of gD protein operably linked to regulatoryelements.

The present invention relates to methods for inhibiting an undesirableimmune response in an individual. The methods comprise administering tothe individual in an amount sufficient to inhibit an undesirable immuneresponse one or more of gD protein, a function fragment of gD protein, anucleic acid encoding gD protein operably linked to regulatory elements,or a nucleic acid encoding a functional fragment of gD protein operablylinked to regulatory elements.

The present invention relates to methods for treating an individual whois about to undergo, is undergoing or has undergone an organ, tissue orcell transplant procedure to prevent rejection and any graft versus hostdisease associated therewith. The methods comprise administering to theindividual in an amount sufficient to down modulate immune responses inthe individual, one or more of gD protein, a function fragment of gDprotein, a nucleic acid encoding gD protein operably linked toregulatory elements, or a nucleic acid encoding a functional fragment ofgD protein operably linked to regulatory elements.

The present invention relates to methods for treating an individual whohas an autoimmune disease. The methods comprise administering to theindividual in an amount sufficient to down modulate immune responses inthe individual, one or more of gD protein, a function fragment of gDprotein, a nucleic acid encoding gD protein operably linked toregulatory elements, or a nucleic acid encoding a functional fragment ofgD protein operably linked to regulatory elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show data from experiments described in the Example in whichimmune responses were compared from animals injected with geneconstructs that encoded an immunogenic protein and a control vector or aconstruct that encoded gD protein.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and intended to refer to proteinaceous compoundsincluding proteins, polypeptides and peptides.

As used herein, gD refers to glycoprotein D from either human herpessimplex virus 1 (HSV-1) or human herpes simplex virus 2 (HSV-2). Inpreferred embodiments, gD is derived from HSV-1.

As used herein, the term “individual” refers to the vertebrate targetedfor use of the present invention. Examples of “individuals” contemplatedby the present invention include but are not limited to humans, higherorder primates, canines, felines, bovines, equines, ovines, porcines,avians, and other mammals.

As used herein, the term “immunogenic vector” relates to a vector whichelicits an immune response. Examples of immunogenic vectors include, butare not limited to viral and bacterial vectors. Some embodiments of thepresent invention relate to methods where the vector administered to theindividual is viral. Examples of viral vectors include but are notlimited to adenovirus, adenovirus associated virus, vaccinia virus, andSV40 virus. In a preferred embodiment, the vector is adenovirus. Someembodiments of the present invention relate to methods where the vectoradministered to the individual is bacterial. Examples of bacterialvectors include but are not limited to Salmonella, mycobacterium and BC.Examples of immunogenic vectors which are useful in gene therapy andwhich can be adapted to the present invention include recombinantadenoviral vectors which are described in U.S. Pat. Nos. 5,756,283 and5,707,618, which are each incorporated herein by reference

As used herein, the term “desired polypeptide” refers to the polypeptidefor which gene therapy is desired. Examples of “desired polypeptides”include human and non-human polypeptides useful as a therapeutic orcompensating protein in gene therapy regimens.

As used herein, the term “therapeutic protein” is meant to refer toproteins whose presence confers a therapeutic benefit to the individual.

As used herein, the term “compensating protein” is meant to refer toproteins whose presence compensates for the absence of a fullyfunctioning endogenously produced protein due to an absent, defective,non-functioning or partially functioning endogenous gene.

In some of the embodiments of the invention that relate to gene therapy,the gene constructs contain either compensating genes or genes thatencode therapeutic proteins. Examples of compensating genes include: thegene which encodes dystrophin, the gene to compensate for the defectivegene in patients suffering from cystic fibrosis, the gene which encodesinsulin, the gene to compensate for the defective gene in patientssuffering from ADA, and the gene encoding Factor VIII. Examples of genesencoding therapeutic proteins include genes which encodeserythropoietin, interferon, LDL receptor, GM-CSF, IL-2, IL-4 and TNF.Additionally, genetic constructs which encode single chain antibodycomponents which specifically bind to toxic substances can beadministered.

In some preferred embodiments, the dystrophin gene is provided as partof a mini-gene and used to treat individuals suffering from musculardystrophy. In some preferred embodiments, a mini-gene which containscoding sequence for a partial dystrophin protein is provided. Dystrophinabnormalities are responsible for both the milder Becker's MuscularDystrophy (BMD) and the severe Duchenne's Muscular Dystrophy (DMD). InBMD dystrophin is made, but it is abnormal in either size and/or amount.The patient is mild to moderately weak. In DMD no protein is made andthe patient is chair-bound by age 13 and usually dies by age 20. In somepatients, particularly those suffering from BMD, partial dystrophinprotein produced by expression of a mini-gene delivered according to thepresent invention can provide improved muscle function.

In some preferred embodiments, genes encoding IL-2, IL-4, interferon orTNF are delivered to tumor cells which are either present or removed andthen reintroduced into an individual. In some embodiments, a geneencoding gamma interferon is administered to an individual sufferingfrom multiple sclerosis.

In some preferred embodiments, the desired polypeptide is encoded by agene encoding human growth hormone.

As used herein, the term “administration” refers to the delivery ofpolypeptides to an individual. “Administration” refers to the deliveryof nucleic acids which encode polypeptides and also the delivery ofpolypeptides to the individual. The term includes, but is not limited todelivery routes including intramuscularly, intravenously, intranasally,intraperatoneally, intradermally, intrathecally, intravenitricularly,subcutaneously, transdermally or topically or by lavage. Modes ofadministration contemplated by this invention include but are notlimited to the use of a syringe, intravenous line, transdermal patch, orneedleless injector.

The present invention provides improved gene therapy vectors that employone of the weapons that the HSV virus uses to evade and undermine aninfected individual's immune system: the gD protein and/or a nucleicacid molecule that encodes it. Armed with this HSV-derived weapon, genetherapy vectors can be made more effective by reducing an individual'simmune response against them. Moreover, the present invention uses theHSV gD protein and/or a nucleic acid molecule that encodes it to treatindividuals who have diseases and conditions associated with undesirableimmune responses.

The present invention arises from the surprising discovery that thedelivery of gD polypeptide suppresses cellular immune responses.Accordingly, when delivered in the context of a gene therapy protocol,gD decreases the immune response directed at the gene therapy vector andcells infected by the same resulting in an increase in the efficacy ofthe gene therapy protocol. When delivered to an individual who has adisease or condition associated with an undesirable immune response suchas an inflammatory or autoimmune disease or tissue or organ transplant,gD decreases the immune response. This immunosuppressive activity of gDmakes it particularly suited for use in gene therapy since codingsequences that encode gD can be included in the gene therapy constructsthemselves. In addition, this immunosuppressive activity of gD makes itan attractive alternative to other immunesuppressive therapies such assteroids in the treatment of autoimmune diseases.

The amino acid sequence of gD and the DNA sequence that encodes gD aredisclosed in Genbank Accession Nos.: E0311—DNA encoding surface proteingD of herpes simplex virus type 1 (HSV-1) Miyarna strain; E03023—DNAencoding surface protein gD of HSV-1; E00402—Herpes simplexvirus-1(HSV-1) glycoprotein D (gD) gene; E00401—Herpes simplex virus-1(HSV-1) glycoprotein D (gD) gene; E00400—Herpes simplex virus-2 (HSV-2)glycoprotein D (gD) gene; E00395—Herpes simplex virus-1 (HSV-1)glycoprotein D (gD) gene; E00394—Herpes simplex virus-2 (HSV-2)glycoprotein D (gD) gene; K01408—Herpes simplex virus type 2 (HSV-2)glycoprotein D (gD-2) gene and flanks; and J02217—HSV1 glycoprotein Dgene, which are each incorporated herein by reference as are thereferences reported with and corresponding to the Genbank Accessionnumbers. U.S. Pat. Nos. 5,958,895, 5,583,028 and 5,955,088, which areeach incorporated herein by reference, disclose isolated gD protein ornucleic acid molecules that encode gD protein.

Functional fragments of gD are those truncated forms of gD protein whichretain immunosuppressive activity. Functional fragments are preferablyabout 5 amino acids, more preferably 10 or more and more preferably 25or more. Functional fragments can be identified routinely by comparingthe immunosuppressive activity of fragments of gD with a negativecontrol. A reduction in immune responses in the presence of a fragmentof gD indicates that the fragment is a functional fragment.

One aspect of the present invention relates to methods of delivering adesired polypeptide to an individual comprising administering to theindividual an immunogenic vector comprising a nucleic acid encoding thedesired polypeptide operably linked to regulatory elements incombination with either the gD polypeptide, or a functional fragmentthereof, or a nucleic acid encoding gD, or a functional fragment thereofoperably linked to regulatory elements, or a combination thereof.According to one aspect of the invention, gD protein or a functionalfragment thereof is delivered to an individual in combination with thedelivery of an immunogenic vector for delivering the coding sequence ofa desired protein in a gene therapy protocol. The gD may be delivered asa protein or a functional fragment thereof or as a nucleic acid moleculewith the coding sequence for gD protein or a functional fragment thereofor any combination thereof. The gD may be delivered in the sameformulation as the gene therapy vector or separately. The gD may bedelivered simultaneously, prior to or subsequent to delivery of the genetherapy vector. In some preferred embodiments, the immunogenic vectorcomprises a nucleic acid molecule with the coding sequence for gDprotein or a functional fragment thereof. In some preferred embodiments,the immunogenic vector comprises gD protein or a functional fragmentthereof. In some preferred embodiments, the immunogenic vector comprisesa nucleic acid molecule with the coding sequence for gD protein and/or afunctional fragment thereof and gD protein and/or a functional fragmentthereof itself. Once delivered to the individual, the nucleic acidencoding the desired polypeptide is expressed and the desiredpolypeptide is synthesized within the individual. The presence of the gDprotein, either delivered as a protein or as a nucleic acid molecule“prodrug” which is expressed inhibits the immune response directed atthe immunogenic vector.

The present invention provides improved gene therapy compositions andmethods. Through gene therapy, polypeptides which are either absent,produced in diminished quantities, or produced in a mutant form in anindividual may be replaced using a vector comprising a nucleic acidencoding the desired polypeptide. The desired polypeptide compensatesfor the lack of the desired polypeptide. Upon administration of thevector to the individual, the individual generates an immune responseagainst the vector. The delivery of gD, either a protein or as a nucleicacid molecule “prodrug”, in combination with the gene therapy vectorthat encodes the desired polypeptide inhibits the immune responsedirected at the immunogenic vector and therefore increases the efficacyof the gene therapy treatment.

The present invention also provides a method of treating individualssuffering from diseases and conditions characterized by undesirableimmune responses such as autoimmune/inflammatory diseases and conditionand organ/tissue/cell transplantation procedures. According to theinvention, methods of treating an individual with a disease or conditionassociated with an undesirable immune response comprise administering tothe individual gD protein or a functional fragment thereof or a nucleicacid encoding gD protein or a functional fragment thereof or acombination of two or more of the same. When a nucleic acid encoding gDprotein or a functional fragment thereof is delivered to an individual,the coding sequence is operably linked to regulatory elements. The gDmay be delivered as a protein or a functional fragment thereof or as anucleic acid molecule with the coding sequence for gD protein or afunctional fragment thereof or any combination thereof. In someembodiments, the gD is delivered as a nucleic acid molecule with thecoding sequence for gD protein and/or a functional fragment thereof. Insome embodiments, the gD and/or a functional fragment thereof isdelivered as a protein. Once delivered to the individual, the presenceof the gD protein, either delivered as a protein or produced by theexpression of the nucleic acid molecule that encodes it, inhibits theundesirable immune response.

According to some embodiments of the present invention, methods areprovided for treating individuals suffering from autoimmune diseases anddisorders. T cell mediated autoimmune diseases include Rheumatoidarthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmunethyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma,polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener'sgranulomatosis, Crohn's disease and ulcerative colitis. Each of thesediseases is characterized by T cell receptors that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases.

According to some embodiments of the present invention, methods areprovided for treating individuals who require immunosuppression such asthose undergoing transplantation procedure including cell, tissue andorgan transplants. In such instances rejection of the transplantedmaterial is reduced and the severity or incidence of side effects suchas graft versus host disease may be lessened.

According to other embodiments, immune suppression can be induced toprevent damage resulting from inflammation. For example, followingspinal cord injuries, a cascade of events leads to inflammation of thespinal cord and surrounding tissues. Use of the present invention mayboth inhibit inflammation of the spinal cord and associated problems andallow delivery of a therapeutic polypeptide to the individual. Forinstance, if an axonal guidance protein is the desired polypeptide, useof the present invention may both inhibit the inflammation of the spinalcord and stimulate axonal regrowth.

As discussed above, in some embodiments, gD is delivered alone and insome embodiments, gD is delivered in combination with a gene therapeuticincluding immunogenic vectors.

In some embodiments of the present invention, a combination of one ormore of gD, a functional fragment thereof, a nucleic acid encoding gD,or a nucleic acid encoding a functional fragment of gD is administeredto a patient.

In some embodiments of the present invention, gD or a functionalfragment thereof is administered as a protein. In some embodiments, thegD or a functional fragment thereof is administered to the individual inthe same formulation as the nucleic acid encoding the desiredpolypeptide. In other embodiments, the gD or a functional fragmentthereof is administered to the individual in a separate formulation thanthe nucleic acid encoding the desired polypeptide. In some embodiments,the formulation containing the gD or a functional fragment thereof isadministered to the individual at the same time as the formulationcontaining the nucleic acid encoding the desired polypeptide. In someembodiments, gD or a functional fragment thereof is delivered as aprotein incorporated within an immunogenic vector.

In some embodiments of the present invention, a nucleic acid thatencodes gD or a functional fragment thereof is administered. In someembodiments of the present invention, the desired polypeptide is encodedby a first nucleic acid while the gD or a functional fragment thereof isencoded by a second nucleic acid. In some embodiments, the nucleic acidthat encodes gD or a functional fragment thereof is administered to theindividual in the same formulation as the nucleic acid encoding thedesired polypeptide. In other embodiments, the nucleic acid that encodesgD or a functional fragment thereof is administered to the individual ina separate formulation than the nucleic acid encoding the desiredpolypeptide. In some embodiments, the formulation containing the nucleicacid that encodes gD or a functional fragment thereof is administered tothe individual at the same time as the formulation containing thenucleic acid encoding the desired polypeptide. In a preferred embodimentof the present invention, the nucleic acid that encodes gD or afunctional fragment thereof and the desired polypeptide are encoded bythe same nucleic acid which is a genome of an immunogenic vector. Insome embodiments, the nucleic acid that encodes gD or a functionalfragment thereof is administered free of an immunogenic vector thatencodes a desired polypeptide.

In some embodiments, the gD coding sequence is delivered separate fromor free of an immunogenic vector. Compositions and methods fordelivering proteins to cells by direct DNA administration have beenreported using a variety of protocols. Examples of such methods aredescribed in U.S. Pat. Nos. 5,593,972, 5,739,118, 5,580,859, 5,589,466,5,703,055, 5,622,712, 5,459,127, 5,676,954, 5,614,503, and PCTApplication PCT/US95/12502, which are each incorporated herein byreference. Compositions and methods for delivering proteins to cells bydirect DNA administration are also described in PCT/US90/01515,PCT/US93/02338, PCT/US93/048131, and PCT/US94/00899, which are eachincorporated herein by reference. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are bothincorporated herein by reference. Nucleic acid molecules can also bedelivered using liposome-mediated DNA transfer such as that which isdescribed in U.S. Pat. Nos. 4,235,871, 4,241,046 and 4,394,448, whichare each incorporated herein by reference.

Formulations comprising an immunogenic vector comprising the nucleicacid having a sequence encoding the desired polypeptide are made upaccording to the mode and site of administration. Such formulation iswell within the skill in the art. In addition to nucleic acids andoptional polypeptides, the formulation may also include buffers,excipients, stabilizers, carriers and diluents.

The pharmaceutical composition comprising gD protein or a fragmentthereof and a pharmaceutically acceptable carrier or diluent may beformulated by one having ordinary skill in the art with compositionsselected depending upon the chosen mode of administration. Suitablepharmaceutical carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, A. Osol, a standard reference textin this field which is incorporated herein by reference.

For parenteral administration, the gD protein can be, for example,formulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Examples of such vehicles are water, saline, Ringer's solution, dextrosesolution, and 5% human serum albumin. Liposomes and nonaqueous vehiclessuch as fixed oils may also be used. The vehicle or lyophilized powdermay contain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

The pharmaceutical compositions comprising gD protein, or fragmentsthereof may be administered by any means that enables the active agentto reach the agent's site of action in the body of a mammal. Becauseproteins are subject to being digested when administered orally,parenteral administration, i.e., intravenous, subcutaneous,intramuscular, would ordinarily be used to optimize absorption.

The dosage administered varies depending upon factors such as:pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment. Usually, adaily dosage of gD protein can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10milligrams per kilogram per day given in divided doses 1 to 6 times aday or in sustained release form is effective to obtain desired results.

Another aspect of the present invention relates to pharmaceuticalcompositions that comprise a nucleic acid molecule that encodes gD and apharmaceutically acceptable carrier or diluent. According to the presentinvention, genetic material that encodes gD protein is delivered to anindividual in an expressible form. The genetic material, DNA or RNA, istaken up by the cells of the individual and expressed. gD that isthereby produced can inhibit immune responses, either those directed atan immunogenic vector or another undesirable immune response such asthose associated with autoimmune and inflammatory disease and conditionsand transplantation procedures. Thus, pharmaceutical compositionscomprising genetic material that encodes gD are useful in the samemanner as pharmaceutical compositions comprising gD protein. gD ornucleic acid molecule with a gD coding sequence may be incorporated intoan immunogenic vector.

Nucleotide sequences that encode gD protein operably linked toregulatory elements necessary for expression in the individual's cellmay be delivered as pharmaceutical compositions using a number ofstrategies which include, but are not limited to, either viral vectorssuch as adenovirus or retrovirus vectors or direct nucleic acidtransfer. Methods of delivering nucleic acids encoding proteins ofinterest using viral vectors are widely reported. A recombinant viralvector such as a retrovirus vector or adenovirus vector is preparedusing routine methods and starting materials. The recombinant viralvector comprises a nucleotide sequence that encodes gD. Such a vector iscombined with a pharmaceutically acceptable carrier or diluent. Theresulting pharmaceutical preparation may be administered to anindividual. Once an individual is infected with the viral vector, gD isproduced in the infected cells.

Alternatively, a molecule which comprises a nucleotide sequence thatencodes gD can be administered as a pharmaceutical composition withoutthe use of infectious vectors. The nucleic acid molecule may be DNA orRNA, preferably DNA. The DNA molecule may be linear or circular, it ispreferably a plasmid. The nucleic acid molecule is combined with apharmaceutically acceptable carrier or diluent.

According to the invention, the pharmaceutical composition comprising anucleic acid sequence that encodes gD protein may be administereddirectly into the individual or delivered ex vivo into removed cells ofthe individual which are reimplanted after administration. By eitherroute, the genetic material is introduced into cells which are presentin the body of the individual. Preferred routes of administrationinclude intramuscular, intraperitoneal, intradermal and subcutaneousinjection. Alternatively, the pharmaceutical composition may beintroduced by various means into cells that are removed from theindividual. Such means include, for example, transfection,electroporation and microprojectile bombardment. After the nucleic acidmolecule is taken up by the cells, they are reimplanted into theindividual.

The pharmaceutical compositions according to this aspect of the presentinvention comprise about 1 ng to 10 mg of nucleic acid in theformulation; in some embodiments, about 0.1 to about 2000 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 1 to about 1000 micrograms of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 1 to about500 micrograms of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 25 to about 250 micrograms of DNA. Mostpreferably, the pharmaceutical compositions contain about 100 microgramsDNA.

The pharmaceutical compositions according to this aspect of the presentinvention are formulated according to the mode of administration to beused. One having ordinary skill in the art can readily formulate anucleic acid molecule that encodes GD. In cases where injection is thechosen mode of administration, a sterile, isotonic, non-pyrogenicformulation is used. Generally, additives for isotonicity can includesodium chloride, dextrose, mannitol, sorbitol and lactose. Isotonicsolutions such as phosphate buffered saline are preferred. Stabilizersinclude gelatin and albumin.

Regulatory elements for nucleic acid expression include promoters,initiation codons, stop codons, and polyadenylation signals. It isnecessary that these regulatory elements be operably linked to thesequence that encodes the desired polypeptides and optionally the GDpolypeptide and that the regulatory elements are operable in theindividual to whom the nucleic acids are administered. For example, theinitiation and termination codons must be in frame with the codingsequence. Promoters and polyadenylation signals used must also befunctional within the cells of the individual.

Examples of promoters useful to practice the present invention includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metallothionein.

Examples of polyadenylation signals useful to practice the presentinvention include but are not limited to SV40 polyadenylation signalsand LTR polyadenylation signals. In particular, the SV40 polyadenylationsignal which is in pCEP4 plasmid (Invitrogen, San Diego Calif.),referred to as the SV40 polyadenylation signal, is used.

EXAMPLE

Animals were immunized by bupivacaine facilitated DNA vaccination usingvarious combinations of gene constructs as shown in FIGS. 1A-1D. In eachof FIGS. 1A, 1C and 1D, co-administration of gD constructs resulted inreduced immune responses against the antigen encoded by the other geneconstruct administered. FIG. 1B shows an anti-gD response. These dataindicate that co-administration of gD-encoding constructs with a secondconstruct that encodes a second immunogenic protein reduces the immuneresponse against the second immunogenic protein.

The foregoing examples are meant to illustrate the invention and are notto be construed to limit the invention in any way. Those skilled in theart will recognize modifications that are within the spirit and scope ofthe invention. All references cited herein are hereby incorporated byreference in their entirety.

1. A method for suppressing an immune response in an individual in needof such treatment comprising identifying said individual andadministering to said individual in an amount sufficient to inhibit anundesirable immune response a nucleic acid molecule encoding gD proteinoperably linked to regulatory elements; wherein said gD protein is HSV-2gD protein.
 2. The method of claim 1 wherein the individual has anautoimmune/inflammatory disease or condition.
 3. A method forsuppressing an immune response in an individual in need of suchtreatment comprising identifying said individual and administering tosaid individual in an amount sufficient to inhibit an undesirable immuneresponse a nucleic acid molecule encoding gD protein operably linked toregulatory elements; wherein said gD protein is selected from the groupconsisting of: HSV-1 gD protein and HSV-2 gD protein, wherein theindividual is undergoing or has undergone a cell, tissue or organtransplant procedure.